Mixed animal feed

ABSTRACT

A mixed animal feed is provided comprising a conventional grass based feed admixed with up to 60% by weight of by-product seed optionally mixed with grape husks. The conventional grass based feed may include lucerne hay. Preferably, the grape seed and, where present, grape husks, have been pre-treated with at least one, and typically a combination of, tannin degrading bacteria that is or are conveniently tannin-hydrolysing lactic acid bacteria. The grape seed would typically be milled prior to contacting it with a suspension of bacteria followed by pelletising the treated grape seed and husks, where present.

FIELD OF THE INVENTION

This invention relates to a mixed animal feed in which there is utilizedan agricultural byproduct. More particularly, but not exclusively, theinvention relates to an animal feed that is suitable for use in feedingsheep.

BACKGROUND TO THE INVENTION

Wheat is the dominant forage available during summer in sheep farmingareas that are subject to a Mediterranean climate, and of particularinterest, in South Africa. Stubble, however, is reported to have lowlevels of nitrogen and available carbohydrates, a high cell wall contentand poor digestibility (Dann and Coombe, 1987), rendering it unsuitableto meet the high nutrient requirements of producing sheep (Aitchinson,1988). It is therefore commonplace to provide supplementary feedingespecially for ewes grazing stubble to provide additional energy andprotein (Aitchinson, 1998; Brand, 1997a). One common form ofsupplementary feed is lucerne hay.

Grapes are widely grown in the Mediterranean area, producingconsiderable quantities of by-products in the form of grape seeds andhusks resulting from the fruit juice and wine producing industries.Traditionally, the grape seeds and husks are dumped or used as compost.

It has now surprisingly been found that a useful mixed animal feed canbe produced using this agricultural by-product.

OBJECT OF THE INVENTION

It is, accordingly, an object of the invention to provide a mixed animalfeed that embodies a proportion of agricultural by-product material.

SUMMARY OF THE INVENTION

In accordance with this invention there is provided a mixed animal feedcomprising a conventional grass based feed admixed with at least oneother animosity constituent, the mixed animal feed being characterisedin that it comprises up to 60% by weight of by-product grape seedoptionally mixed with grape husks.

Further features of the invention provide for the conventional grassbased feed to be lucerne hay, typically in pelleted or otheragglomerated form; for the grape seed and, where present, grape husks,to be pre-treated with at least one, and typically a combination oftannin degrading bacteria, preferably tannin-hydrolysing lactic acidbacteria; for the grape seeds and any grape husks admixed therewith tobe dried, milled, typically in a hammer mill and pelleted for admixturewith the conventional grass based feed; and for the milled by-product,in instances in which it is to be pre-treated with bacteria, to besuspended in a suspension that is inoculated with the bacteria prior todrying and pelletising.

Regarding suitable bacteria to be employed for the aforesaid purpose, anumber of bacteria capable of degrading tannins have been identified,viz. Streptococcus bovis, Streptococcus caprinus and Streptococcusgallolyticus (Brooker et al., 1994; Ossawa et al., 1995; Sly et al.,1997). Many of the strains were isolated from the rumen of goatsbrowsing on Acacia (rich in tannins). The strains were resistant tocondensed tannins from Acacia anuera and grew in media containingconcentrations as high as 2.5%, w/v (Brooker et al., 1994).

Despite the identification of diverse populations of tannin tolerantbacteria from a number of animals, e.g. goat (Brooker et al., 1994;McSweeny et al., 1996), koalas (Osawa, 1990; 1992; Osawa and Sly, 1992)and other ruminants (Nelson et al., 1995; Odenyo and Osuji, 1998),little is known about the relationships these organisms have with other(normal) gut microflora and the mechanisms they use to degrade tannins(Brooker, 2000).

The invention is therefore based on the fact that the basal diet ofsheep, consisting of lucerne hay, can be altered by replacing variousproportions, and up to about one half (50%) of the lucerne hay, withgrape seeds and husks. The invention, in its preferred implementation isstill further based on the fact that binding of tannins (from the grapeseeds and husks) to proteins can be decreased by treating the grapeseeds and husks beforehand with a combination of tannin-hydrolyzinglactic acid bacteria.

The invention therefore also provides a feed constituent comprisingby-product grape seed optionally mixed with grape husks and wherein thegrape seeds and any husks mixed therewith have been treated with atannin degrading bacteria. The tannin degrading bacteria is preferablyat least one and typically a mixture of tannin hydrolysing bacteria. Theby-product grape seed and any husks mixed therewith are preferablymilled and sized, typically to a size of about 1 mm in diameter prior tocontacting with an aqueous culture of the relevant bacteria.

In order that the invention may be more fully understood an extendeddescription thereof with reference to the drawings and the results ofvarious investigations that have been carried out to date, now follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:—

FIG. 1 is a bar chart illustrating the comparative activity of thetannin hydrolysing bacteria identified as TS1 TS2 TS3 and TS4 herein ascompared to a control batch utilizing a control Lactobacillus sp. thatcould grow in the suspension and that tested slightly positive fortannin hydrolysis;

FIG. 2 is a set of four graphs illustrating the hydrolysis of tannins ina tannin rich medium in respect of each of the four bacteria identifiedas TS1 (FIG. 2A), TS2 (FIG. 2B), TS3 (FIG. 2C) and TS4 (FIG. 2D)individually; and,

FIG. 3 is a flow diagram illustrating a proposed treatment of grape pipsand husks preparatory to forming the mixed feed.

DETAILED DESCRIPTION OF VARIOUS ASPECTS OF THE INVENTION

A breakdown of the protein, fat and various fibre contents of the seeds,and a combination of husks and seeds, of a mixture of four red winecultivars (Merlot, Shiraz, Carignan and Cabernet Sauvignon), ispresented in Table 1 (see page 14). The amino acid composition of thelatter is presented in Table 2 (see page 14). In each instancecomparisons were made with plant products that are normally added toanimal feed.

The overall chemical composition of grape pips and a combination ofhusks and pips was very similar (Table 1). The protein content of thehusks and pips was lower than that recorded for Alfalfa (12.7% versus15%). However, the fat content of the grape pips was much highercompared to Alfalfa (10.3 and 7.9% versus 1.6%). The higher fat contentwould result in an increase in energy production and is, in the light ofthis, considered to be an advantage. The higher ADF (acid detergentfiber) and NDF (neutral detergent fiber) contents recorded in grape pipsand husks would, however, slow down the enzymatic conversion of theanimal feed in the gut and is thus considered to be a disadvantage. Thetotal DM (dry matter) is also set out in Table 1.

The grape seeds contained a larger variety of amino acids than wasrecorded for maize and soya (Table 2), rendering it a more suitableanimal feed. However, Lysine, a limiting and very important amino acidin animal feed, is present at very low concentrations (0.387%). Thisindicated that a mixed feed could work effectively.

Tannins are not easily degradable. Metabolic energy in animal feed isderived mostly from starches, sugars, carbohydrates, fats and oils.Binding of tannins to any of the latter substrates is believed torestrict the digestibility of the substrate (Tangendjaja, 2000), whichin turn may lead to a lowering in the digestibility of the substrates inan animal feed. Furthermore, binding of tannins to proteins is believedto the produce insoluble or soluble tannin-protein (and alsotannin-enzyme) complexes which, when ingested, may lead to a lowering ofenzyme activity, followed by a decrease in intestinal metabolic activitywhich may lead to malnutrition.

Tannins occur in red grapes, and are present either in hydrolysable orcondensed forms (Butler, 1989). There is an inverse relationship betweenhigh tannin level in forage and palatability, digestibility andvoluntary intake. Grape seeds (pips), -husks and -skins are rich incondensed tannin content (approximately 14 g STE, sorghum TanninEquivalents, per kg dry mass).

In the light of this it was determined that the basal diet of sheep,consisting of lucerne hay, can be altered by replacing up to one half(50%) of the lucerne hay with grape seeds and husks.

Grape seeds and husks of Merlot, Shiraz, Carignan, Cabernet Sauvignonwere dried, pooled in equal amounts by weight, mixed and pelleted. Thebasal diet, pelleted lucerne hay, was then supplemented for testpurposes with the grape seeds and husks such that the latter contributed0, 12.5, 25.0, 37.5 and 50.0% of the total dry matter intake.

Twenty Dohne merino ram lambs (41.4±2.3 kg) were used in a voluntaryintake and digestion trail. A completely randomized design was used andthe animals were assigned to five diets consisting of 0, 12.5, 25.0,37.5 and 50% grape seeds and husks.

The chemical composition of the five diets, and the grape seeds andhusks, before being fed to the animals, is listed in Table 3 (see page15). The protein content decreased as the percentage grape seeds andhusks increased in the diet, while the CP-ADF (protein attached to cellwalls, and therefore indigestible) increased. The level of condensedtannin increased dramatically as the percentage grape seeds and husksincreased in the diets. The condensed tannins are reported to bind toproteins and sometimes may reduce the protein digestibility (Walton etal., 2001).

All the animals were vaccinated and drenched before the experiments, andwere kept in individual pens. Feeding was ad lib and at a level close tomaintenance (40-45 g DM kg⁻¹LW^(0.75) per day), as recommended by Van Esand Van der Meer (1980). During the trail, which lasted 35 days (14 daysfor acclimatization and 21 days for the experiment), daily water and drymatter intake were measured. Faeces were collected daily from eachanimal, dried at 50° C. for 96 h, and ground through a 1 mm screen.

The fecal, orts and feed samples were analyzed for dry matter (DM), ash,crude protein (CP) and ether extract (EE) according to AOAC (1984)methods. To determine neutral detergent fiber (NDF) and acid detergentfiber (ADF) the methods proposed by Van Soest et al. (1991) werefollowed. Acid-detergent insoluble nitrogen (ADIN) was measured (Licitraet al., 1996), and the results reported as crude protein (ADF-CP). Thesorghum tannin equivalent method was used for determination of condensedtannins.

Blood samples (10 ml) were taken from each sheep at the end of thedigestibility trial. Blood was taken from the jugular vein intoheparinized tubes and centrifuged for 20 min at 3 000 rpm (revolutionsper minute) to separate the plasma, which was stored at −20° C. Theplasma was analyzed according to normal procedures used for diagnosingdomestic animal hepatic and kidney damage and general disorders (Kaneko,1989). Components measured were total protein, plasma urea nitrogen andcreatinine. In addition, the plasma enzymes aspartate aminotransferase(AST) and gamma glutamyltranspeptidase (GGT) were measured.

The average feed intake, water intake and blood metabolic profile dataare listed in Table 4 (see page 16).

According to Table 4 the voluntary feed and water intake were notsignificantly influenced (P≧0.05) by the percentage grape seeds andhusks included in the diet. The final body weight was also notnegatively influenced (P≧0.05) by the inclusion of grape seeds (pips)and husks up to 50% of the diet. The presence of tannin in a forage hasbeen assumed to affect voluntary intake (McLeod, 1974). However, in thistrial intake problems were not observed with inclusion levels of up to50% of diet dry matter.

There were no differences between diets in any plasma metabolite, exceptfor blood urea nitrogen (Table 4). An increase of creatinine can berelated with renal failure, but the level found in the present studyfell within the normal range for sheep (Kaneko, 1989). No significantchanges in plasma enzymes AST and GGT were found. These enzymes are usedto detect if tannin-related hepatotoxicity occurred (Zhu & Filippish,1992). In sheep fed with lucerne hay, the blood urea nitrogenconcentration was higher (P≦0.05) than in sheep fed with the dietsincluding the grape seeds and husks, which is directly related to thelimitation in protein digestibility in sheep fed husks and pips. Similarresults were found by Silanikove et al. (1996) where urea concentrationwas higher in goats fed with tannin-rich leaves than when fed wheatstraw.

The digestibility of the five diets is shown in Table 5 (see page 16).

According to this data, grape seeds and husks could be considered lowquality roughage. The digestibility of the crude protein, neutraldetergent fiber and acid detergent fiber decreased significantly, whilethe dry matter digestibility showed a strong tendency towards a lowerdigestibility as the percentage grape seeds and husks increased in thediet. This result may be due to several factors. Firstly, the husks andpips had a much higher level of CP-ADF (crude protein bound to theindigestible fiber fraction) than that of lucerne hay (71.5% vs. 17.6%of the total protein is bound to the fiber). Secondly, it could be dueto the presence of condensed tannins in husks and pips. The condensedtannin content of the husks and pips was 20 times higher than that oflucerne hay. These compounds may form complexes with proteins andcarbohydrates (Makkar et al., 1996), decreasing the available proteinand energy for rumen microorganisms.

The decrease in diet digestibility as the percentage husks and pipsincluded in the diet increased, might be due to factors such as highlevels of proteins bound to the acid detergent fiber and condensedtannins. However, sheep accepted an inclusion up to 50% in the diet andtoxic effects were not evident in this study.

It is therefore not exactly clear as to whether or not the effects ofthe tannins will adversely affect the mixed feed according to theinvention but, nevertheless, an attempt was made to diminish theseeffects utilizing tannin-hydrolysing lactic acid bacteria.

Isolation of Tannin Hydrolysing Strains:

Tannin-hydrolysing lactic acid bacteria were isolated from the faeces ofgoats and sheep. Fecal samples were streaked onto MRS Agar (Merck)plates, incubated for 3-5 days at 37° C., and colonies of variousmorphology selected. A total of 200 isolates were collected and testedfor tannin hydrolysis as follows:

BHI (Brain Heart Infusion) Agar (Merck), supplemented with 0.5% yeastextract (Unilab), was overlaid with 5 ml of a 2% (w/v) tannic acid(Unilab, 5944000) solution and left at room temperature (approx. 25° C.)for at least one hour. The excess tannic acid solution was then decantedand rinsed from the plates by using sterile distilled water. The plateswere left to dry and then inoculated with 100 μl of an active growingculture from BHI broth. The plates were incubated at 37° C. for at least24 h.

From the above plates, six colonies tested positive for the degradationof tannic acid (observed as clearing zones surrounding the colonies).Pure cultures were obtained by repeated streaking onto BHI Agar andstores at −80° C. in 40% (v/v) glycerol. Four strains with the highesttannin hydrolysis activity, based on the reactions recorded on the BHIAgar plates, were selected.

Identification and Characteristics of the Strains:

Identification was done by using the API 50CHL carbohydrate fermentationprofile test system. Two of the strains were identified as Streptococcusspp. and two as Lactobacillus spp. The strains were numbered TS1 and TS2(streptococci) and TL1 and TL2 (lactobacilli).

Characteristics of the Strains:

-   Gram-positive, catalase negative. Cocci in chains (streptococci) or    elongated cells (lactobacilli).-   None of the strains could utilize tannic acid as a sole carbon    source.-   L-lactic acid is produced from the fermentation of glucose.-   Gucose, starch, cellobiose, gallactose, mannose, trehalose, sucrose,    lactose, fructose, maltose, raffinose and inulin are fermented.    Rhamnose, glycerol, xylose, sorbitol, inositol and arabinose are not    fermented.-   Optimal growth at 37° C.-   Good growth in the absence of CO₂.-   Growth in MRS broth, but prefers BHI broth.    Treatment of the Grape Seeds and Husks:

The dried grape seeds and husks were milled in a hammer mill to aparticle size of 1 mm in diameter. Three parts of sterile distilledwater were added to one part of the milled grape seeds. Peptone (2%,w/w) was added to the grape seed suspension and then heat-treated for 2min at 100° C. The heated suspension was left to cool down to roomtemperature (approx. 25° C.). One of the batches was inoculated with 10%(v/v) of an equal combination of strains TS1, TS2, TL1 and TL2. Theother batch was inoculated with a control Lactobacillus sp. that couldgrow in the suspension, but tested slight positive for tanninhydrolysis.

Treatment of the grape seed (pip) and husk suspension withtannin-hydrolyzing bacteria resulted in the hydrolysis (“splitting”) oftannins from the protein (peptone added to the suspension). A clearincrease in free tannins, as determined with a standard acid butanol andspectrophotometric assay, was recorded in the batch treated with thetannin-hydrolyzing bacteria (FIG. 1). The highest level of free tannins(OD=0.2835) was recorded after 11 days of treatment with thetannin-hydrolyzing bacteria. The control batch revealed much lowerhydrolytic activity (FIG. 1).

Binding of tannins to proteins can be decreased by treating the grapeseeds and husks beforehand with a combination of tannin-hydrolyzinglactic acid bacteria (TS1, TS2, TL1 and TL2).

In a repeat of the above study, but with the four strains usedseparately, the hydrolysis of tannins in a tannin-rich medium wasdetected over a period of 10 days by using the acid-butanol assay. FIG.2 indicates the percentage hydrolysis by each of the lactic acidbacteria. Of all the strains, Streptococcus ST1 (FIG. 2A) andStreptococcus ST2 (FIG. 2B) had the highest degree of tannin hydrolysis(approx. 25 and 35%, respectively). Lactobacillus strains LT1 (FIG. 2C),LT2 (FIG. 2D) also had the ability to hydrolise tannins, but to a lesserextent (approx. 10 and 20%, respectively). As indicated in FIG. 2,maximal tannin hydrolysis occurred at day 8 (day 6 for LT2), followed bya decrease in the percentage hydrolysis.

In vitro tests are currently being done to determine the digestibilityof the treated grape seed (pip) and husk suspension. The methoddescribed by Tilley et al. (1963) will be used.

Strains TS1, TS2, TL1 and TL2 may be cultured in 10% (v/v) molasses (pH7.0) for 24 h before treating of the grape seeds, husks and pips asmentioned above.

As shown in FIG. 3, a proposed process for the pretreatment of grapepips and husks to be used according to the invention could involve theaddition of say 10 litres of a suspension of the mixed culture that isstored at 4° C. in a 10% v/v molasses solution in a storage container(1) to 90 litres of a sterile 10% v/v molasses solution that has beensterilised typically by boiling for 15 minutes that is supplied from astorage container (2). The mixture is fermented in a fermentation vessel(3) for 24 hours at a temperature of 30-37° C. Fermentation is carriedout without aeration and with slow stirring. 90 litres of the resultantsuspension is then added to 910 litres of the sterile molasses solutionin a further fermentation vessel (4) and fermentation takes place asindicated above. Part or all of the resultant suspension can then beadded to pre-treated, milled grape seed and husk at the rate of threeparts of suspension to one part of milled grape seed and husk in asuitable tumble type of apparatus (5) in which it is tumble slowly at atemperature of 25 to 30° C. without aeration. Any balance of thissuspension be stored in a storage tank (6) at 4° C.

The pre-treated, milled grape seed and husk, is preferably passedthrough a hammer mill (7) to a particle size of 1 mm in diameter andheat treated at 70° C. for 30 minutes in a suitable kiln (8).

The thoroughly mixed, is then drum or spray dried as indicated bynumeral (9) to a moisture content of about 15 percent preparatory topelletising as indicated by numeral (10) and subsequent mixing with theconventional grass based feed as indicated at (11).

Probiotic Properties:

Antimicrobial Resistance.

Grazing animals are usually treated with the antibiotics ampicillin,chloramphenicol, neomycin, furazolidone, streptomycin, tetracycline,kanamycin, nalidixic acid, gentamycin, ciprofloxacin, sulphonamides,erythromycin, and oxacilin to prevent infectious diseases caused by thepathogens Escherichia coli, Salmonella, Staphylococcus aureas,Campylobacter and enterococci (Barton, Pratt and Hart, 2003). StrainsTS1, TS2, TL1 and TL2 were resistant to most of the antibiotics tested(Table 6 (see page 17)). However, the antibiotics erythromycin,tetracycline, novobiocin, ampicillin and chloramphenicol resulted intotal growth inhibition.

Growth at Different pH Values.

To determine if the lactic acid bacteria were able to function as aprobiotic in grazing animals, their growth was tested at different pHvalues. All the strains were able to grow between pH 5 and 8 in BHIbroth. The best growth was obtained between pH 7 and 8. Initial mediumpH of 4 and below completely inhibited growth of the four strains.

Resistance to Bile Salts.

Strains TS1, TS2, TL1 and TL2 grew in the presence of 9% (w/v) bilesalt, but with an increase in lag phase. This indicated that thebacteria do have the ability to adapt to high bile salt concentrationsas would be found in the intestine of most grazing animals.

Attachment to Intestinal Cells.

Bacligh™ studies were done to determine if the four bacteria would beable to attach to intestinal cells. Streptococcus strains TS1 and TS2attached to the mucus layer of porcine ileum, whereas strains TL1 andTL2 were unable to do so, or attached only slightly. Attachment to mucusincreases the possibility of the cells becoming colonized in theintestine.

Whilst it is not yet clear as to the long-term effects of utilizinggrape seeds and husks that has not been treated to relieve the effectsof the high tannin content as a part of an animal feed typicallycontaining as the other part lucerne hay it is expected that the treatedgreat seeds and husks will in any event be highly useful as a part ofsuch feed and will put to good use an agricultural byproduct that doesnot find any particular present use.

Tables: TABLE 1 Chemical composition of unpressed and pressed grapeseeds (pips) Chemical component (%) Grape pips Husks and pips AlfalfaADF 52.4 40.8 35 NDF 58.2 42.7 38.5 DM 90.8 94 89 ASH 2.3 — 7.9 Protein8.2 12.7 15 Fat 10.3 7.9 1.6 Fiber 35.7 21.9 28

TABLE 2 Amino acid content (%) of the grape seeds (pips) Amino acidGrape pips Maize Soya Amino Acid Rec 8.656 Aspartic acid 0.758 Threonine0.225 2.13 1.82 Serine 0.365 Glutamic acid 2.040 Proline 0.564 Glycine0.840 Alanine 0.360 Valine 0.415 1 2.36 Methionine 0.034 1.51 0.63Isoleucine 0.355 2.72 2.28 Leucine 0.565 10.38 3.55 Tyrosine 0.181Pheynylalanine 0.337 3.94 2.36 Histidine 0.226 1.3 1.23 Lysine 0.3871.07 2.89 Arginine 0.546 2 3.45 Ammonia 0.680

TABLE 3 Physical (on an air dry basis) composition (%) and chemical (ona dry matter basis) composition (%) of the experimental diets Grape pipsExperimental diet 1 2 3 4 5 & husks Lucerne hay 100 87.5 75.0 62.5 50Grape pips and husks 0 12.5 25.0 37.5 50 Chemical composition Dry matter92.5 92.1 92.0 92.0 92.4 92.3 Organic matter 88.9 89.4 89.1 90.7 92.092.9 Ash 11.1 10.6 10.9 9.3 8.0 7.1 Crude protein 18.2 17.9 17.2 16.215.1 13.7 Neutral detergent fibre 44.0 43.4 43.8 43.9 43.6 43.3 Aciddetergent fibre 33.4 34.5 36.3 37.0 38.5 43.4 CP-ADF (g/100 g CP¹) 3.24.3 4.8 5.7 5.5 9.8 Ether extract (fat) 2.4 3.4 4.6 6.9 7.2 11.0 Totalcondensed tannins, 0.7 2.4 4.1 5.7 7.4 14.1 gSTE²/kg DM¹CP = Crude protein²STE = Sorghum Tannin Equivalents

TABLE 4 Average feed, water intake and blood metabolic profile of sheepfed the different diets Lucerne hay:Grape seeds (pips) and husks Item100:0 87.5:12.5 75:25 62.5:37.5 50:50 SEM P Initial body weight, kg 41.741.4 41.4 41.5 40.8 2.25 0.99 Final body weight, kg 45.0 44.6 43.3 43.841.5 2.40 0.85 DM intake, g/day 1840 1916 1881 1943 1952 148.5 0.98 DMintake/W^(0.75), g/day 110 116 114 117 119 7.08 0.94 Water intake, l/day7.74 7.49 7.67 6.84 6.19 0.77 0.58 Water intake/W⁰⁷⁵, l/day 0.46 Bloodurea nitrogen, 9.6^(a) 7.4^(b) 7.5^(b) 6.4^(b) 6.7^(b) 0.73 0.05 mg/100ml Total protein, mg/100 ml 68.0 67.5 69.8 71.3 68.3 2.36 0.79Creatinine, mg/100 ml 118.8 122.0 119.3 125.3 124.8 5.50 0.87 AST,units/l 75.8 93.3 73.5 80.5 89.8 6.25 0.25 GGT, units/l 80.5 70.0 72.076.0 68.5 4.26 0.31^(a,b,c)Values in rows bearing different superscript letters showssignificant (P ≦ 0.05) differencesSEM = standard error of the mean;P = probability

TABLE 5 Apparent digestion coefficients of the diets Lucerne hay:Grapeseeds (pips) and husks Item 100:0 87.5:12.5 75:25 62.5:37.5 50:50 SEM PApparent digestibility (%) Dry matter 57.1 53.9 50.4 46.8 48.0 2.450.053 Crude protein 68.4^(a) 64.2^(ab) 59.0^(b) 56.3^(bc) 52.5^(c) 1.910.0002 Neutral detergent fibre 42.6^(a) 33.5^(b) 32.3^(b) 23.9^(c)18.5^(c) 2.12 <0.0001 Acid detergent fibre 39.9^(a) 26.4^(b) 23.7^(b)c17.1^(cd) 12.8^(d) 3.21 0.0003 Ether extract (fat) 39.3^(a) 66.1^(b)77.7^(c) 82.6^(c) 79.0^(c) 3.82 <0.0001^(a,b,c)Values in rows bearing different superscript letters showssignificant (P ≦ 0.05) differencesSEM = standard error of the mean;P = probability

TABLE 6 Antibiotic resistance of the our lactic acid bacteriaAntibiotics TS1 TS2 LT1 LT2 Amikacin (30 μg/disc) C C C C Ampicillin (10μg/disc) A A A A Bacitracin (10 units/disc) C C C C Cefepime (30μg/disc) C C C C Ceftriaxone (30 μg/disc) C C C C Ceftazidime (30μg/disc) C C C C Cephazolin (30 μg/disc) C C C C Cefotaxine (30 μg/disc)C C C C Cefuraxime (30 μg/disc) C C C C Chloramphenicol (30 μg/disc) A AA A Ciprofloxacin (5 μg/disc) B B B B Compound Sulphonamides (300μg/disc) C C C C Colistin sulphate (25 μg/disc) C C C C Cloxacillin (5μg/disc) C C C C Clindamycin (10 μg/disc) C C C C Erythromycin (15μg/disc) A A A A Fusidic acid (5 μg/disc) B B A A Furazolidone (50μg/disc) C C C C Gentamicin (10 μg/disc) C C C C Kanamycin (30 μg/disc)C C C C Metronidazole (5 μg/disc) C C C C Methicillin (5 μg/disc) C C CC Neomycin (30 μg/disc) C C C C Novobiocin (5 μg/disc) A A A ANitrofurantoin (300 μg/disc) B B B B Nystatin (100 units/disc) C C C CNalidixic acid (30 μg/disc) C C C C Oflaxacin (5 μg/disc) B B B BOxacillin (1 μg/disc) C C C C PolymyxinB (300 units/disc) C C C CRifampicin (5 μg/disc) B B B B Sulphamethoxazole (100 μg/disc) C C C CStreptomycin (10 μg/disc) C C C C Tetracyclin (30 μg/disc) A A A ATobramycin (10 μg/disc) C C C CDiameter of inhibition zones: 17 mm and more (A), between 12 and 16 mm(B), no zones to 11 mm in diameter (C).

REFERENCES

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1. A mixed animal feed comprising a conventional grass based feedadmixed with at least one other animosity constituent comprising up to60% by weight of by-product grape seed optionally mixed with grapehusks.
 2. A mixed animal feed as claimed in claim 1 in which theconventional grass based feed includes lucerne hay.
 3. A mixed animalfeed as claimed in claim 1 in which the grape seed and, where present,grape husks, have been pre-treated with at least one tannin degradingbacteria.
 4. A mixed animal feed as claimed in claim 3 in which thegrape seed and, where present, grape husks, have been pre-treated with acombination of tannin degrading bacteria.
 5. A mixed animal feed asclaimed in claim 3 in which at least one of the bacteria is atannin-hydrolysing lactic acid bacteria.
 6. A mixed animal feed asclaimed in claim 3 in which the bacteria includes one or more ofStreptococcus bovis, Streptococcus caprinus and Streptococcusgallolyticus.
 7. A mixed animal feed as claimed in claim 3 in whichpre-treatment with bacteria was effected by suspending the grape seedand, where present, grape husks in a suspension that is inoculated withthe bacteria prior to drying.
 8. A mixed animal feed as claimed in claim1 in which the grape seeds and any grape husks admixed therewith havebeen dried, milled and pelleted for admixture with the conventionalgrass based feed.
 9. An animal feed constituent characterised in that itcomprises by-product grape seed optionally mixed with grape huskswherein the grape seeds and any husks mixed therewith have been treatedwith a tannin degrading bacteria.
 10. An animal feed constituent asclaimed in claim 9 in which the tannin degrading bacteria is at leastone tannin hydrolysing bacteria.
 11. An animal feed constituent asclaimed in claim 9 in which the by-product grape seed and any husksmixed therewith are milled and sized, typically to a size of about 1 mmin diameter prior to contacting with an aqueous culture of the relevantbacteria.
 12. An animal feed constituent as claimed in claim 9 in whichthe constituent is in a pelletised form.